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2 CHAPTER 6 Choice of the donor according to HLA typing and stem cell source 1. Introduction Allogeneic haematopoietic stem cell transplantation (allohsct) is widely used to treat patients with malignant and nonmalignant haematological disorders. Initially, the principal source of HSCs was bone marrow (BM) from an HLAidentical sibling for transplantation in children and young adults. Subsequently, the choice of donors and the sources of HSCs have enlarged, extending transplant indications to more patients, especially adults. Today, transplant physicians must choose among stem cell sources between bone marrow, granulocyte colonystimulating factor (G CSF)mobilised peripheral blood stem cells (PBSC), or umbilical cord blood (UCB). The donor can be an HLA identical sibling, an matched unrelated donor (MUD), a haploidentical family peripheral blood stem cell (PBSC) or bone marrow donor or, an HLAmismatched unrelated UCB donor. 2. Use of different cell sources for HSCT in Europe Baldomero et al. performed an EBMT survey of HSCT activity in 2009 and analysed the trends over the previous 5 years (1, 2). They collected reports of 31,322 HSCT from 624 centres from 43 countries of which 28,033 were first transplants (41% allogeneic and 59% autologous). The main indications were leukaemias (31%; 92% allogeneic), lymphomas (58%; 12% allogeneic), solid tumours (5%; 6% allogeneic) and nonmalignant disorders (6%; 88% allogeneic). For allogeneic HSCT there were more unrelated than HLAidentical sibling donors (51% vs 43%). The proportion of peripheral blood as stem cell source was 99% for autologous and 71% for allogeneic HSCT. Unrelated cord blood was used in 756 cases (7%). For further details, see the EBMT activity survey at Figure 1 shows World Marrow Donor Association (WMDA) data on the use of different stem cell sources worldwide. 3. Choice of the stem cell source Traditionally, haematopoietic stem cells were harvested from the posterior iliac crests under general anaesthesia. More recently, mobilised peripheral blood stem cells (PBSC) have been increasingly used in both auto and allohsct. Mobilisation of haematopoietic stem cells to the peripheral blood in sufficient numbers can be achieved by the classical administration of growth factors such as GCSF (allohsct) and/or myelosuppressive chemotherapy (autohsct). Unmanipulated cord blood (CB) cells collected and cryopreserved at birth have been used both in related and unrelated HLAmatched and mismatched allogeneic transplants in children, and more recently in adults. Comparison of the yield of various cell sources is given in Table 1. In all cases, safety of the donor is the major concern meaning that the precollection workup should be particularly meticulous, and should be performed by HAEMATOPOIETIC STEM CELL TRANSPLANTATION 91

3 Figure 1: Number of BM, PBSC and CBU donations according to continents Number of BM and PBSC donations 6,000 5,000 4,000 3,000 2,000 1, Year Africa Asia Australia Europe North America South America Number of CBUs provided 1,600 1,400 1,200 1, Year Africa Asia Australia Europe North America South America WMDA data a trained experienced doctor. Informed consent must be fully and deeply explained, and all the risks must be discussed. In some countries, this medical examination is performed by a different medical team (e.g. anaesthetists). All possible medical contraindications must be looked for and no risk should be taken however great is the need of the patient. In some countries a special procedure is applied for child donors. It is a requirement to establish a central reporting system to collect adverse events and longterm followup of the donors (3, 4). 92 THE EBMT HANDBOOK 2012 EDITION

4 CHAPTER 6 Choice of the donor according to HLA typing and stem cell source Table 1 : Number of cells according to stem cell source Volume collected Med CD34 content Med CD3 content Target cell dose Bone marrow ml/kg 2 3 x 10 6 /kg* 25 x 10 6 /kg >2 x 10 8 TNC/kg Peripheral blood ml 8 x 10 6 /kg 250 x 10 6 /kg 5 10 x 10 6 CD34 + /kg Umbilical cord blood ml 0.2 x 10 6 /kg 2.5 x 10 6 /kg >3 x 10 7 TNC/kg *per kg recipient body weight 3.1 HLA identical sibling bone marrow transplant When available, this is the preferred source of HSCs; typically bone marrow cells are harvested under general anaesthesia in the posterior iliac crest. There are no contraindications related to age; collections have been performed at all ages from a few months to elderly adults. As long as general anaesthesia can be given. The yield is better in young children and adults and decreases with age. The volume of collection must be adjusted to the donor and recipient weight but should not exceed ml/kg donor body weight. Collection teams should not aspirate a volume more than 5 ml for each aspiration to avoid dilution of the bone marrow with blood. After collection, the bone marrow is filtered and volume reduced and is then infused iv directly to the patient. 3.2 Peripheral blood stem cell transplant GCSF mobilised PBSC have been recently used more frequently for HLA identical sibling transplants. The indications for using PBSC rather than BM are not really standardised, but many factors are involved: availability of operating room, lack of personnel to perform bone marrow aspiration, contraindication to general anaesthesia and choice of the donor. Most teams avoid collecting PBSCs in young children because of possible side effects of GCSF or problems of venous access. According to NMDP, 76% of adult unrelated donors donated GCSF mobilised PBSC. Severe but rare adverse effects have been observed including splenic rupture and sickle cell crisis. There are no definitive conclusions regarding the longterm effects of GCSF treatment on healthy donors (4). More recently plerixafor, a novel small molecule antagonist, which reversibly inhibits the interaction of SDF1 and CXCR4, has been used in poor mobilisers with or without GCSF. It has not been tested in healthy donors (5). Recently biosimilars of GCSF appeared in the market. In view of the absence of longterm studies their use is not recommended in healthy normal donors (6). 3.3 Haploidentical related HSCT donors The number of related haploidentical donors is increasing, but there are very few large comparative or randomised studies (7, 8). HAEMATOPOIETIC STEM CELL TRANSPLANTATION 93

5 Historically, the first haploidentical transplants pioneered by the Perugia group utilised GCSF mobilised Tcell depleted CD34 + cells. More recently, interesting preliminary data have been reported with new protocols using unmanipulated bone marrow cells or marrow harvested after in vivo GCSF, together with high dose immunosuppression or cyclophosphamide (9 12). 3.4 Matched unrelated HSCT donors The number of matched unrelated donor transplants has been increasing worldwide, with more than 18 million adult donors registered in Bone Marrow Donor Worldwide (BMDW; This collection of HLA phenotypes from bone marrow donor registries and cord blood banks give the opportunity to transplant physicians from around the world to perform preliminary searches through the internet for a preliminary view of the availability of potential suitable donors (Figure 1 and Figure 2). The annual report of the WMDA ( is a product of the Donor Registries Figure 2: Number of bone marrow and cord blood units worldwide 300 Umbilical cord blood units, n= 450,000 Thousands Millions Adult marrow donors, n= 14,000,000 From WMDA and BMDW 94 THE EBMT HANDBOOK 2012 EDITION

6 CHAPTER 6 Choice of the donor according to HLA typing and stem cell source Working Group whose mission is to develop guidelines for donor recruitment and maintenance of donor confidentiality, track the efficiency of donor searches and develop consensus standards for the logistics of transporting stem cell products across international borders (13, 14). The standards by which stem cell products are collected and transported result from the collaborative efforts of several organisations represented within the Worldwide Network for Blood and Marrow Transplantation ( Most often the donors are chosen on the basis of high resolution allelic typing for HLAA, B, C, DRB1, DQB1, DPB1. The best choice is a 9/10 or 10/10 donor. In the case of several potential donors, it is better to recruit a donor who is male, young, ABO matched and CMV negative if the recipient is negative or CMV positive if the recipient is positive. The choice between bone marrow and PBSC depends on donor choice, centre preference and indication for HSCT. It is known that using PBSC gives more GvHD which is favourable in malignant diseases where increasing GvHD decreases the risk of relapse while in nonmalignant diseases, especially in aplastic anaemia, it decreases survival (15). 3.5 Cord blood Cord blood bank organisation Since the first human cord blood transplant, performed in 1988, cord blood banks have been established worldwide for collection and cryopreservation of cord blood for allogeneic haematopoietic stem cell transplant (16). Today, a global network of cord blood banks and transplant centres has been established and provides a common inventory. Several studies have shown that the number of cells is the most important factor for engraftment, while some degree of HLA mismatch is acceptable (16, 17). The absence of ethical concern and the unlimited supply of cells explains the increasing interest in using cord blood for developing regenerative medicine. Since the first UCBT, more than 20,000 CBT have been reported worldwide and more than 600,000 cord blood units have been stored in more than 100 cord blood banks ( (18). The main practical advantages of using cord blood as an alternative source of stem cells are the relative ease of procurement, the absence of risk for mothers and donors, the reduced likelihood of transmitting infections, particularly CMV, and the ability to store fully tested and HLA typed potential transplants in the frozen state, available for immediate use. Eurocord was established in 1995; its principal objectives were to collect outcomes data provided by cord blood banks and transplant centres. Eurocord has collected, from 1988 to October 2010, 6736 UCBT from Europe and transplant centres in other countries. Five hundred and ninetysix transplants have been reported using HAEMATOPOIETIC STEM CELL TRANSPLANTATION 95

7 related donors (majority HLA identical sibling donors), mainly for children with malignant and nonmalignant disorders and 6140 have been performed in the unrelated transplant setting for children (n=3287) and adults (n=2770). In order to promote education and information, Eurocord has launched a new European program entitled: an Online CME program in cord blood technology and transplantation for providing a learning tool on the scientific, technical, clinical, regulatory aspects of cord blood, easily accessible at a time and language convenient for users ( Based on this international cooperation Eurocord has published crucial reports which have been the basis of the rapid development of cord blood transplant. Netcord ( was created in 1998 to establish good practices in umbilical cord blood storage, facilitate donor search, improve the quality of the grafts, standardise excellence criteria on an international scale and importantly establish procedures for bank accreditation. The inventory of Netcord, the cooperative network of large experienced UCB banks, currently has more than 300,000 cryopreserved UCB units ready for clinical use for unrelated recipients and more than 8,624 grafts shipped. In the USA, the National Marrow Donor Program (NMDP) has established a similar cord blood bank network. Collaborations between Netcord Eurocord and NMDP have been established with the goal to provide the most appropriate and high quality cord blood unit for a specific patient. A summary of cord blood banks activity is available at (18). National regulatory agencies and transplant centres are aware of the need of international standards whose major objectives are to promote quality throughout all phases of cord blood banking with the goal to achieve consistent production of high quality placental and umbilical cord blood units for transplantation. All these aspects are detailed in the last version of the NetcordFACT Standards ( As the number of cord blood units is increasing, it appeared that it was necessary to improve the quality of the units for costefficient management of the banks. The optimal number of cord blood units is not really known but should approach 9 per 10,000 inhabitants. Most banks prefer to bank only the largest units of more than 70 ml in order to obtain at least 3 x 10 7 nucleated cells/kg recipient bodyweight Criteria for cord blood donor choice In several studies including Eurocord, neutrophil and platelet recovery were associated with the degree of HLA mismatch, the number of total nucleated cells (TNC) collected and infused and the use of GCSF after transplant. Coexistence of HLA class I and II disparities and high CD34 + cell dose in the graft were associated with only with more severe GvHD grade IIIIV. Disease relapse was higher in matched transplants, showing a graft versus leukaemia effect (17 20). 96 THE EBMT HANDBOOK 2012 EDITION

8 CHAPTER 6 Choice of the donor according to HLA typing and stem cell source General considerations: Current HLA definition in cord blood banks is based on HLA serological typing for A and B and allelic typing for HLADRB1. Avoid cord blood units with 3 or 4 HLA disparities. At selection, diagnosis and presence of patient HLA antibodies against the HLA antigens of the cord blood unit should be considered. HLA compatibility appears to be more important for patients with nonmalignant disorders than for those with malignant disorders. If the criterion for the minimum number of cells for a single UCB transplantation is not achieved, a double cord blood transplantation should be considered. CFUGM and viability are generally not used for donor selection. Cell dose requirements must increase with the number of HLA mismatches. Cord blood units with 6/6 or 5/6 HLA match. HLAA or HLAB mismatches are preferable to DRB1 mismatches. In malignant disorders: Nucleated cell dose: at freezing, the cell dose should be >2.5 to 3.0 x 10 7 /kg after thawing, the cell dose should be >2.0 to 2.5 x 10 7 /kg. CD34 + cell dose: at freezing or after thawing, the dose should be approximately 1.2 to 1.7 x 10 5 /kg. Based on Eurocord unpublished and preliminary data, it seems likely that HLA DRB1 mismatch may lead to high graftversusleukaemia (GvL) effect in patients transplanted in nonremission (but there is also a greater risk of GvHD). In nonmalignant disorders: Higher total and CD34 + cell dose is requested, and HLA match is preferable. Cord blood units with 4/6 HLA match. HLAA or HLAB mismatches are better than HLADRB1 mismatches. In malignant disorders: Nucleated cell dose: at freezing >3.5 x 10 7 /kg after thawing >3.0 x 10 7 /kg. CD34 + cell dose: at freezing or after thawing, approximately >1.7 x 10 5 /kg HLADRB1 mismatch may lead to a high GvL effect in advanced phase of the diseases. In nonmalignant disorders: Nucleated cell dose: at freezing, minimum cell dose 4 to 5 x 10 7 /kg HAEMATOPOIETIC STEM CELL TRANSPLANTATION 97

9 after thawing, minimum 3.5 x 10 7 /kg. CD34 + cell dose: no available data, but should be higher than 2 to 2.5 x 10 5 /kg. CB units with 3/6 HLA match. Should be avoided, but in extremely severe cases may be considered for patients with malignant disorders; a high nucleated cell dose should be given. Not recommended for patients with nonmalignant disorders. Other considerations: If several cord blood units are available that fit the above criteria, the following should be taken into consideration: Cord blood bank accreditation status and location ABO compatibility Allele HLA typing of HLAA and B Other HLA factors such as HLAC, high resolution HLA typing, NIMA; KIR are under study and cannot be used at this stage for donor choice. Double cord blood transplants can be recommended if the cell dose is insufficient with a single CBU. The total dose of both combined units should be at least 3 x 10 7 TNC/kg. HLA matching between the 2 units and the recipient must be as matched as possible but no firm recommendation can be given. Note that double cord transplant gives a good engraftment and survival with more GvHD and less relapse than single cord blood transplant. 4. Comparison between stem cell sources and algorithm for donor search 4.1 Bone marrow compared to PBSC An EBMTinitiated prospective randomised trial that involved 350 patients with standardrisk leukaemia compared HLA identical sibling BM to PBSC and showed that the incidence of grades IIIV acute GvHD was significantly higher in patients who underwent PBSCT. There was a higher incidence of chronic GvHD after PBSCT (21). On the other hand, there were no significant differences in overall survival or leukaemiafree survival between the BMT and PBSCT groups at the 3year followup (5). A large retrospective International Bone Marrow Transplant Registry (IBMTR) and EBMT registry analysis involving 824 patients revealed the following data: the incidence of chronic GvHD at 1 year was significantly higher in patients after PBSCT, and treatmentrelated mortality rates and leukaemiafree survival rates were in favour of PBSCT in patients in advanced stages of leukaemia. A later retrospective EBMT 98 THE EBMT HANDBOOK 2012 EDITION

10 CHAPTER 6 Choice of the donor according to HLA typing and stem cell source registry analysis of 3465 adult patients with AML and ALL again revealed a higher incidence of chronic GvHD after PBSCT. However, rates of acute GvHD, leukaemiafree survival and overall survival were similar after BMT and PBSCT (5). In aplastic anaemia, 3 studies with HLA identical siblings or matched unrelated donors have shown that PBSC gave more GvHD and decreased survival (22, 23). Despite these results the number of PBSCT is increasing steadily. There are many possible reasons, including preference of the centres, since the logistics of organising PBSC collection are easier than organising bone marrow collection with general anesthaesia. One main reason advocated by the centres is donor safety, but a recent report on over 50,000 donations for PBSC in Europe documented a significantly higher rate of severe adverse events with PBSC collection, per 10,000 donations for PBSC compared with 4.32 per 10,000 donations for BM. Fatalities were also higher, though not significantly, in PBSC versus BM donors (4 versus 1 fatality) (5). 4.2 HLA identical siblings versus matched unrelated transplants To determine whether the risks of allogeneic transplantation are different when the donor is a fully matched unrelated donor based on 10/10 HLA alleles compared to an HLAidentical sibling, Woolfrey et al. performed a retrospective analysis of 1448 patients with highrisk or advanced haematologic malignancies given T replete grafts after myeloablative conditioning. No statistically differences were found between the two groups in survival, diseasefree survival and nonrelapse mortality for patients with highrisk disease or those given bone marrow as a graft source. However, for patients with intermediaterisk disease receiving peripheral blood grafts, they observed higher nonrelapse mortality and lower overall survival in the 10/10 MUD compared to matched sibling donors (24). A prospective French study showed no statistically significant difference between MUD and HLA matched sibling donors. The Essen group performed the same study in 101 patients who were given either a 10/10 MUD or a HLA identical sibling transplant and did not find any difference in survival. In both studies MUD transplant gave more GvHD. These studies show that use of a HLA 10/10 MUD is a good option when an HLA identical sibling donor is unavailable. A higher degree of HLA mismatches, 9 or 8/10, is often used but there are no strong data to suggest what mismatches are acceptable. 4.3 Cord blood versus unrelated bone marrow transplant In children, with malignant diseases, two studies have compared the outcome of unrelated UCBT and BMT (25, 26). Eurocord published a study comparing the outcome of matched unrelated BMT (HLA 6 out of 6), either unmanipulated or T depleted, with that of mismatched UCBT. Results showed that after UCBT, engraftment was delayed, GvHD was reduced similarly to Tcell depleted BMT and there was no HAEMATOPOIETIC STEM CELL TRANSPLANTATION 99

11 difference in relapse or in leukaemiafree survival. Eapen et al. for the CIBMTR and the New York Cord Blood Bank (NYCBB) compared the outcomes of 503 children with acute leukaemia given an unrelated mismatched UCBT with 282 unrelated BM transplant recipients. HLA allelemismatched BM recipients had more acute and chronic GvHD without decreasing leukaemiafree survival (LFS). Importantly, they found that even using an allelematched BM donor, LFS was not statistically different from one or 2 HLA disparate UCBT and that an HLAmatched UCBT recipient had better outcomes compared to HLA allelematched BM recipients. However, an increased transplant related mortality was observed in children transplanted with a low CB cell dose (<3 x 10 7 /kg) and a 1 HLA disparate CB graft or in children given a 2 HLA disparate UCBT, independently of the cell dose infused. Interestingly, use of 2 HLA mismatched UCBT was associated with a lower incidence of relapse (27). The same studies were performed in adults with malignancies. Eurocord compared adults with acute leukaemia receiving either a matched unrelated bone marrow transplant (HLA 6 out of 6) or a mismatched cord blood transplant. Results showed that, despite a delay of engraftment, UCBT gave a similar leukaemiafree survival to BMT. In the same issue of the journal, CIBMTR and NYCBB showed that, in adults with malignancies, UCBT gave the same LFS survival as 1 antigen mismatched unrelated bone marrow transplant (UBMT) (25, 26). At the same time, a Japanese study showed that CBT gave better results than matched unrelated bone marrow transplants. In a metaanalysis, combining these published studies, 161 children and 316 adults undergoing UCBT, along with 316 children and 996 adults undergoing UBMT were analysed. Tcell depleted UBMT was excluded where data were available, and only fully matched UBMT was used in the analysis. Pooled comparisons of studies of UCBT and UBMT in children found that the incidence of chronic GvHD was lower with UCBT, but the incidence of grade III IV acute GvHD did not differ. There was no difference in 2year overall survival in children when studies were pooled. For adults, transplantationrelated mortality (TRM) and LFS were not statistically different. Recently, Eurocord and CIBMTR performed a study comparing the outcome of unrelated HLAmatched or 12 antigen mismatched bone marrow (n=364) or GCSF mobilised peripheral blood (n=728) with that of mismatched cord blood transplant (n=148) in adults with acute leukaemia. In multivariate analysis, TRM was higher after UCBT, but relapse rate and GvHD were lower, resulting in the same LFS compared to the other sources of stem cells (Figure 3) (28, 29). The results of these comparative studies and the metaanalysis (30), gathered together, showed that UCBT is feasible in adults when a cord blood unit contains a high number of cells and should be considered an option as an allogeneic stem cell source for patients lacking a HLAmatched bone marrow donor; despite increased HLA disparity, UCB 100 THE EBMT HANDBOOK 2012 EDITION

12 CHAPTER 6 Choice of the donor according to HLA typing and stem cell source Figure 3: Leukaemiafree survival according to stem cell source Adjusted probability, % Leukaemiafree survival BM matched, 41% PBPC matched, 39% 70 UCB, 33% PBPC mismatched, 34% 30 BM mismatched, 34% Months Reproduced with permission from (29) from unrelated donors offers sufficiently promising results compared with matched UBMT in adults with haematological malignancies leading to the conclusion, as in children, that the donor search process for BM and UCB from unrelated donors should be started simultaneously, especially in patients with acute leukaemia, where the time factor is crucial (30, 31). Further improvement has been obtained by the use of double cord blood transplant and of reduced intensity conditioning regimens (31). These papers were the hallmark of the worldwide development of cord blood transplant as they clearly demonstrated that cord blood transplant could be used in adults as well as in children, and furthermore showed that unrelated mismatched cord blood transplant gave the same results as a HLA matched unrelated bone marrow transplant. 4.4 Related haploidentical transplants compared to other sources of hematopoietic stem cells There are very few publications on the longterm results of haploidentical related transplants. Although the technology has changed over the years, large randomised studies are needed. Retrospective comparative studies show that early results are comparable to other sources of cells but longterm results are not available. Leung et al. evaluated 190 children with very highrisk leukaemia who underwent allogeneic haematopoietic cell transplantation in two sequential treatment eras to determine HAEMATOPOIETIC STEM CELL TRANSPLANTATION 101

13 whether those treated with contemporary protocols had a lower risk of relapse or toxic death and whether nonhla identical transplantations yielded poorer outcomes (32). For the recent cohorts, 5year overall survival rates were 65% for the 37 patients with ALL and 74% for patients with AML. These rates compared favourably with those of earlier cohorts (28% and 34% respectively). The recent improvement was observed regardless of the cell source (5yr OS for matched sibling transplants 70 versus 24%, MUD 61 versus 37% and haploidentical 88 versus 19%). This improvement was attributable to a reduction in infections, in regimenrelated toxicity and in leukaemiarelated death. Survival probability was dependent on leukaemia status and minimal residual disease. The authors conclude that with the improvement of results over time, bone marrow transplantation should be considered for all children with very highrisk leukaemia regardless of matched donor availability. 5. Algorithm of donor choice and conclusion (see Figure 4) Results of allogeneic HSCT have been markedly improving over time, due to a better expertise of the centres, better supportive care and better prevention and treatment of infections. Figure 4: Algorithm of donor search Patient and family HLA typing HLA identical sibling: HSCT No HLA identical sibling: Allele typing for A, B, C, DRB1, DQB1, DPB1 Search for an unrelated donor in bone marrow registries and cord blood banks HLA 9/10 or 10/10 matched unrelated donor: HSCT Unrelated cord blood 3 x 10 7 TNC/kg and 1 2 HLA MM: HSCT Related haploidentical: HSCT If transplant urgent: prefer cord blood or related haploidentical transplant If not enough cells in a single cord blood unit: consider double cord Consider other factors: indication of the transplant, ABO, CMV, donor sex Expertise of the centres is very important for donor selection for HLA mismatched transplants 102 THE EBMT HANDBOOK 2012 EDITION

17 b) Splenic rupture c) Haematoma d) All of the above The following characteristics are considered when selecting a cord blood unit for transplantation: a) Allelelevel HLA typing at class I and II b) Cell viability c) Total nucleated cell dose per kilogram of patient body weight d) b + c A 35 year old gentleman with acute lymphoblastic leukemia experiences an ontherapy bone marrow relapse. He achieves a second complete remission after reinduction chemotherapy. He does not have a matched sibling and you decide the best treatment option for this gentleman is an unrelated donor transplantation. He has an adult unrelated donor who is HLAmatched at A, B, C, DRB1 and DP1. He also has a cord blood unit with adequate total nucleated cell dose/kg and mismatched at 1locus (5/6 HLAmatched). The waiting period to procure the adult donor graft is 3 months. Which of the donor sources would you choose for this patient? a) Matched unrelated adult donor b) Mismatched cord blood unit Compared to bone marrow, peripheral blood stem cell transplant gives: a) Better engraftment b) Less GvHD c) More relapse d) Better survival THE EBMT HANDBOOK 2012 EDITION

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